Oncogenesis is considered to be a multi-stage process involving introduction of alterations in the DNA which, if left unrepaired, often lead to mutagenic changes in the DNA of daughter cells after cell division (1-3). Accumulation of mutagenic damage in genomic DNA can ultimately lead to the necessary and sufficient conditions for malignant conversion and tumorigenesis. Diverse agents have been implicated in carcinogenesis, and lead to introduction of DNA modifications with mutagenic potential. Interest has focused on oxidative damage from single electron oxidative steps induced by multiple forms of reactive oxygen species (ROS) resulting from such in vivo exposures.
Modification of DNA bases by the introduction of a single oxygen atom in a purine ring is a type of single-electron oxidation caused by ROS. Such DNA base damage includes the production of 8-hydroxydeoxyguanine (8-hydroxy-Gua) and 8-hydroxydeoxyadenine (8-hydroxy-Ade). Studies indicate that these lesions are frequently present in substantial concentrations in the DNA in cancerous tumors or in histologically normal tissue from cancer patients, as high as 1 in 102 or 103 normal bases, but generally very low in normal healthy tissues. Studies have shown that there is a 1- to 2% level of misreading of 8-hydroxy derivatives during DNA replication resulting in base substitutions and transversions (4,5).
Analysis of single electron oxidations of adenine in solution has demonstrated that the redox status of the reaction controls the structure of the reaction product (6). Under oxidative conditions, the transient 8-oxo-Ade radical is quantitatively converted through the loss of an electron and protonation to form the 8-OH-Ade product, the structure of which is shown in FIG. 1B. Alternatively, under reductive conditions, an electron is added and after protonation and possible rearrangement the ring-opening formamidopyrimidine, 4,6-diamino-5-formamidopyridine (also referred to herein as FAPY-A, FAPY-Ade, or FAPY-adenine) derivative is the exclusive product. The structure of FAPY-Ade is shown in FIG. 1A. Recent studies have shown FAPY derivatives of purine bases to also be mutagenic (7,8). In particular, FAPY-A was shown to lead to A→C transversions and FAPY-G to G→T transversions (7).
Analysis of ROS-induced DNA base lesions present in normal human breast tissue, breast cancer tumors, and histologically normal breast tissue from breast cancer patients have been conducted using gas chromatography-mass spectrometry (GC-MS) (9). Two general types of results were obtained. Increased levels of 8-hydroxy purine derivatives were observed in both cancerous tumors and the surrounding normal tissue from cancer patients compared to normal, non-cancer specimens. In contrast, substantial elevations of the ring-opening FAPY derivatives were highly expressed in normal tissues but were very low in cancer derived tissues. This qualitative difference in the nature of ROS-induced DNA damage results from the fundamentally different redox status of cancerous or pre-cancerous tissues (oxidative) versus the more reductive environment of normal tissues (10-13). Because of their accumulation in cancerous tissues, 8-OH-derivatives of purines have been utilized as markers for carcinogenesis. However, redox chemistry suggests they may only be detectable in more oxidative tissues occurring in cancer and later stages of carcinogenesis. Thus, the alternate FAPY-derivatives may have greater utility in analyzing the earlier stages of carcinogenesis and potentially offer a useful risk assessment marker for predicting future cancer incidence. In particular, FAPY derivatives may have more mutagenic significance in early stages of carcinogenesis in normal tissues than 8-hydroxy derivatives whose expression is high in more oxidative conditions such as cancerous tissues and tumors.
To date, most studies focusing on effects of ROS on DNA have relied on chemical approaches for detection and quantitation. These include GC-MS/SIM (14-18) and high performance liquid chromatography-electrochemical detection (HPLC-ECD) (19-21) methodologies that require initial purification of tissue DNA in high purity, are time consuming, cumbersome, and not practical for applied diagnostic or screening uses outside of a research laboratory.